Formulation comprising a gemcitabine-prodrug

ABSTRACT

This invention relates to pharmaceutical formulations of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, a monophosphate derivative of the well-known oncology drug gemcitabine. In particular, the invention relates to formulations which comprise a polar aprotic solvent, preferably dimethyl acetamide (DMA). Formulations comprising these solvent provide therapeutically effective treatments of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The invention also relates to methods of using said formulations and kits comprising said formulations.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/010,338, filed Sep. 2, 2020; which is a continuation of U.S. patentapplication Ser. No. 16/142,948, filed Sep. 26, 2018, now U.S. Pat. No.10,786,523; which is a continuation of U.S. patent application Ser. No.15/308,475, filed Nov. 2, 2016, now U.S. Pat. No. 10,117,888; which is a§ 371 national stage application based on Patent Cooperation TreatyApplication serial number PCT/GB2015/051858, filed Jun. 25, 2015; whichclaims the benefit of priority to GB 1411253.6, filed Jun. 25, 2014; IN2050/MUM/2014, filed Jun. 25, 2014; and GB 1417646.5, filed Oct. 6,2014.

This invention relates to pharmaceutical formulations ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (chemical name:2′-Deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzoxy-L-alaninyl)]phosphate), a monophosphate derivative of thewell-known oncology drug gemcitabine. In particular, the inventionrelates to formulations which comprise a polar aprotic solvent,preferably dimethyl acetamide (DMA). Formulations comprising thesesolvents provide therapeutically effective treatments ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The formulations ofthe invention may be diluted to the required concentration shortlybefore administration.

BACKGROUND

Gemcitabine (1; marketed as Gemzar®) is an effective nucleoside analoguethat is currently approved to treat breast, non-small cell lung, ovarianand pancreatic cancers and widely used to treat a variety of othercancers including bladder, biliary, colorectal and lymphoma.

Gemcitabine's clinical utility is limited by a number of inherent andacquired resistance mechanisms. At the cellular level resistance isdependent on three parameters: (i) the down-regulation of deoxycytidinekinase, necessary for the activation into the phosphorylated moiety;(ii) the reduced expression of nucleoside transporters, in particular,hENT1 required for uptake by cancer cells; and (iii) the up-regulationof catalytic enzymes especially cytidine deaminase that degradesgemcitabine.

WO2005/012327 describes a series of phosphate derivatives of gemcitabineand related nucleoside drug molecules. Among themgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2) isidentified as a particularly effective compound. These compounds appearto avoid many of the inherent and acquired resistance mechanisms whichlimit the utility of gemcitabine (‘Application of Pro Tide Technology toGemcitabine: A Successful Approach to Overcome the Key Cancer ResistanceMechanisms Leads to a New Agent (NUC-1031) in Clinical Development’;Slusarczyk et all; J. Med. Chem.; 2014, 57, 1531-1542).

Unfortunately, NUC-1031 is extremely lipophillic and thus poorly watersoluble (by calculation: <0.1 mg/mL), and the ionisable moieties,pyrimidine nitrogen and phenolic hydroxyl, have calculated pKa valueswhich lie out-side the pH range suitable for parenteral administration.It is essentially insoluble in water, regardless of salt content or pH,and this has serious implications for the development of clinicallyacceptable methods for delivering the compound at sufficiently highdosages for effective treatment. Sometimes, the delivery of drugmolecules as lipophillic as NUC-1031 can be achieved but only with anunacceptable level of pain to the patient.

NUC-1031 exists as a mixture of two diastereoisomers, epimeric at thephosphate centre:

It is an aim of certain embodiments of this invention to provide apharmaceutical formulation ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate which delivers aneffective dose.

It is an aim of certain embodiments of this invention to provide astable pharmaceutical formulation ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. For intravenousadministration, suitable infusion formulations typically should bestable for greater than 30 minutes and up to 48 hours. Typically, forintravenous administration the formulation should be stable both toprecipitation of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate andto degradation of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

It is an aim of certain embodiments of this invention to provide apharmaceutical formulation ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate which delivers aneffective dose intravenously.

It is an aim of certain embodiments of this invention to provide aparenteral formulation ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate which can beadministered in either a peripheral vein or via a central line. Thus, itis an aim of certain embodiments of this invention to provide aformulation which has an osmolarity which is acceptable foradministration via a peripheral vein.

Certain embodiments of this invention satisfy some or all of the aboveaims.

BRIEF SUMMARY OF THE DISCLOSURE

In accordance with a first aspect of the present invention there isprovided a pharmaceutical formulation comprising:

gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate;

a polar aprotic solvent; and

optionally one or more pharmaceutically acceptable excipients.

The polar aprotic solvent may be selected from dimethylacetamide (DMA)dimethylsulfoxide (DMSO) and N-methypyrrolidone (NMP). Preferably, thepolar aprotic solvent is DMA. DMA offers the best solubility profile ofthose tested.

The polar aprotic solvent (e.g. DMA, DMSO or NMP) may be pharmaceuticalgrade. The polar aprotic solvent (e.g. DMA) may be the administrationvehicle or it may be that the formulation is diluted before use with anadministration vehicle which provides desirable characteristics. Thus,the formulation may be ready for infusion and have the polar aproticsolvent (e.g. DMA) as a major component; or it may be a formulationwhich has the polar aprotic solvent (e.g. DMA) as a major component andis intended to be diluted before administration to generate aformulation which is ready for infusion and has the polar aproticsolvent (e.g. DMA) only as a minor component; or it may be a formulationwhich is ready for infusion, has the polar aprotic solvent (e.g. DMA)only as a minor component and results from the dilution of a formulationin which polar aprotic solvent (e.g. DMA) is a major component. Thus,the polar aprotic solvent (e.g. DMA) may represent from 0.1% v/v to 100%v/v of the formulation.

Very few pharmaceutically acceptable solvents dissolve sufficientquantities of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate todeliver a therapeutically effective dose intravenously. Of those thatdo, many are not stable, i.e. thegemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate will tend toprecipitate out of solution. The inventors have surprisingly found thatsolvents which do generate a stable solution are generally polar aproticsolvents, for example DMA, DMSO and NMP. Of those solvents that havebeen found to be capable of dissolvinggemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, the inventors havefound that certain polar aprotic solvents, and in particular DMA, areparticularly able to hold it in solution at a concentration necessary todeliver the required dose when that solution is diluted with an aqueousvehicle. Thus, the use of polar aprotic solvents, and in particular DMA,provides a twofold advantage over other formulation solvents which,surprisingly, makes it an excellent medium for deliveringgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate to patients in apractical and therapeutically effective manner.

The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate may be present asa mixture of phosphate diastereoisomers or it may be present as the(S)-epimer or as the (R)-epimer in substantially diastereomerically pureform. ‘Substantially diastereomerically pure’ is defined for thepurposes of this invention as a diastereomeric purity of greater thanabout 90%. If present as a substantially diastereoisomerically pureform, the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate may have adiastereoisomeric purity of greater than 95%, 98%, 99%, or even 99.5%.

The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate may be present asa mixture of phosphate diastereoisomers. Administering NUC-1031 as amixture of diastereoisomers thus offers a practical and economic methodof delivering an effective treatment. Non clinical evidence suggeststhat there is no difference in biological effectiveness between the twoisomers.

Alternatively, the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate 2may be present as the (S)-epimer 3 in substantially diastereomericallypure form. The (S)-epimer shows a surprising and remarkable increase insolubility relative to the (R)-epimer which allows more convenientformulation, increases the stability of the formulation and reduces therisk of precipitation in the giving sets or central line. It may alsoallow the drug to be delivered in such a way as to reduce patientdiscomfort when administered via a peripheral vein in a dilutedformulation.

The formulation of the invention may be for dilution by a predeterminedamount shortly before administration, i.e. up to 48 hours (e.g. up to24, 12 or 2 hours) before administration.

The formulation may also comprise one or more pharmaceuticallyacceptable solubilizers, e.g. a pharmaceutically acceptable non-ionicsolubilizers. Solubilizers may also be called surfactants. Illustrativesolubilizers include polyethoxylated fatty acids and fatty acid estersand mixtures thereof. Suitable solubilizers include polyethoxylatedcastor oil (e.g. that sold under the trade name Kolliphor® ELP); orpolyethoxylated stearic acid (e.g. that sold under the trade namesSolutol® or Kolliphor® HS15); or polyethoxylated (e.g. polyoxyethylene(20)) sorbitan monooleate, (e.g. that sold under the trade name Tween®80).

In certain preferred embodiments, the formulation comprises more thanone pharmaceutically acceptable solubilizer.

The formulation may also comprise an aqueous vehicle. The formulation ofthe invention may be ready to administer, in which case it willtypically comprise an aqueous vehicle.

The formulation may be for parenteral, e.g. for intravenous,subcutaneous or intramuscular administration. Preferably, theformulation is for intravenous administration. The administration may bethrough a central vein or it may be through a peripheral vein.

The total dose of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate ina formulation suitable for administration will typically be from 250 mgto 3 g, e.g. from 1 g to 2 g, e.g. about 1.5 g.

It may be that the polar aprotic solvent (e.g. DMA) represents 30% ormore by volume of the formulation. Thus, it may be that the polaraprotic solvent (e.g. DMA) represents 50% or more, e.g. 60% or more byvolume of the formulation. The polar aprotic solvent (e.g. DMA) mayrepresent 95% or less by volume of the formulation, e.g. 90% or less.The formulation may also comprise an aqueous vehicle (e.g. saline). Theaqueous vehicle may be present in 50% or less by volume of theformulation, e.g. 30% or less by volume of the formulation. Typicallythe aqueous vehicle (e.g. saline) will represent 5% or more, e.g. 10% ormore, by volume of the formulation.

It may be that the concentration of thegemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in the formulationsolvent(s) is 500 mg or less per mL. It may be that the concentration100 mg or more per mL. Preferably, the concentration is from 200 mg to300 mg, e.g. from 225 mg to 275 mg, e.g. about 250 mg, per mL.

Certain preferred formulations comprise:

-   -   from 30% to 95% by volume DMA;    -   from 5% to 50% by volume aqueous vehicle; and    -   from 100 mg to 400 mg (e.g. from 100 mg to 300 mg) per mL        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

More preferred formulations comprise:

-   -   from 70% to 90% by volume DMA;    -   from 10% to 30% by volume aqueous vehicle (e.g. saline); and    -   from 200 mg to 300 mg per mL        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.        The formulations described in the previous four paragraphs, in        which the polar aprotic solvent (e.g. DMA) is present as a major        component, may, for example, be used for administering        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in the form        of a mixture of phosphate diastereoisomers. They can also be        used to administer        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in the form        of the (S)-phosphate epimer in substantially diastereomerically        pure form. The formulations described in these paragraphs can be        used by administering (e.g. by infusion or injection) the        formulation without it being diluted prior to administration.        They may be administered through a central vein.

Alternatively, these formulations may be diluted to form a formulationsuitable for administration through a peripheral vein.

It may be that the polar aprotic solvent (e.g. DMA) represents 10% ormore, e.g. 20% or more by volume of the formulation. Thus, it may bethat the polar aprotic solvent (e.g. DMA) represents 80% or less, e.g.60% or less by volume of the formulation. The polar aprotic solvent(e.g. DMA) may represent 40% or less by volume of the formulation. Theformulation may also comprise one or more solubilizers (e.g. one or morepolyethoxylated fatty acids). The one or more solubilizers may represent90% or less by volume of the formulation, e.g. 80% or less by volume ofthe formulation. Typically the one or more solubilizers will represent30% or more, e.g. 50% or more or 60% or more, by volume of theformulation. One preferred formulation comprises the drug as a solutionin a 30%:70% DMA:solubilizer mixture.

It may be that the concentration of thegemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in the formulationsolvent(s) is 200 mg or less per mL, e.g. 150 mg or less or 120 mg orless. It may be that the concentration is 40 mg or more per mL, e.g. 60mg or more. Preferably, the concentration is from 70 mg to 110 mg, e.g.about 75 mg or about 100 mg, per mL.

Certain preferred formulations comprise:

-   -   from 20% to 80% by volume DMA;    -   from 30% to 80% by volume solubilizer or solubilizers; and    -   from 50 mg to 150 mg per mL        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The        formulation may also comprise an aqueous vehicle, e.g. in an        amount from 1% to 15% by volume.

Certain particularly preferred formulations comprise:

from 20% to 80% by volume DMA;

from 20% to 60% by volume a first solubilizer;

from 5% to 40% by volume a second solubilizer;

from 2% to 12% an aqueous vehicle; and

from 50 mg to 150 mg per mLgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The firstsolubilizer may be a polyethoxylated castor oil (e.g. that sold underthe trade name Kolliphor® ELP). The second solubilizer may be apolyethoxylated sorbitan monooleate (e.g. that sold under the trade nameTween® 80). The formulation may also comprise an aqueous vehicle, e.g.in an amount from 3% to 15% by volume.

The formulation may comprise:

-   -   from 50% to 60% by volume DMA;    -   from 20% to 30% by volume the first solubilizer;    -   from 8% to 15% by volume the second solubilizer;    -   from 4% to 10% an aqueous vehicle; and    -   from 75 mg to 125 mg per mL        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

The formulations described in the previous five paragraphs, in which thepolar aprotic solvent (e.g. DMA) is present as a major component, can beused, for example, for administeringgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in the form of the(S)-phosphate epimer in substantially diastereomerically pure form. Theycan also be used for administering a mixture of R and S epimers or the Repimer. The formulations described in these paragraphs are typicallydiluted with an aqueous vehicle prior to administration. Once diluted,they may be administered through a peripheral vein.

These formulations may be formed by diluting a formulation that does notcontain any solubilizers.Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate can degrade in thepresence of certain solubilizers.

It may be that the polar aprotic solvent (e.g. DMA) represents 0.1% ormore, e.g. 0.5% or more or 1% or more by volume of the formulation.Thus, it may be that DMA represents 10% or less, e.g. 5% or less or 3%or less by volume of the formulation. The polar aprotic solvent (e.g.DMA) may represent 8% or less or 2% or less by volume of theformulation. The formulation may also comprise an aqueous vehicle (e.g.WFI). The aqueous vehicle may be present in 99.5% or less by volume ofthe formulation, e.g. 99% or 98% or less by volume of the formulation.Typically the aqueous vehicle will represent 85% or more, e.g. 90% ormore or 95% or more, by volume of the formulation. The formulation mayalso comprise one or more solubilizers (e.g. one or more polyethoxylatedfatty acids). The one or more solubilizers may represent in 10% or lessby volume of the formulation, e.g. 7.5% or less or 5% or less or 3% orless by volume of the formulation. Typically the one or moresolubilizers will represent 0.1% or more, e.g. 0.5% or more or 1% ormore or 2% or more, by volume of the formulation.

It may be that the concentration of thegemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in the formulationsolvent(s) is 12.0 mg or less per mL or 10.0 mg or less per mL, e.g. 7.0mg or less or 4.5 mg or less per mL. It may be that the concentration is1.0 mg or more per mL, e.g. 2.0 mg or more. Preferably, theconcentration is from 2.5 mg to 11 mg per mL, e.g. from 3 mg to 7 mg permL, e.g. about 4.5 mg per mL.

Certain preferred formulations comprise:

-   -   from 0.1% to 15% (e.g. 0.5 to 5%) by volume DMA;    -   from 0.1% to 15% (e.g. 0.1% to 7.5%) by volume solubilizer or        solubilizers;    -   from 85% to 99% by volume aqueous vehicle; and    -   from 2.0 mg to 12.0 mg (e.g. from 2.0 mg to 10.0 mg) per mL        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

Certain particularly preferred formulations comprise:

-   -   from 0.5% to 10% by volume DMA;    -   from 0.2% to 4% by volume a first solubilizer;    -   from 0.1% to 2% by volume a second solubilizer;    -   from 85% to 99% by volume aqueous vehicle; and    -   from 2.0 mg to 12.0 mg (e.g. from 2.0 mg to 10.0 mg) per mL        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The first        solubilizer may be a polyethoxylated castor oil (e.g. that sold        under the trade name Kolliphor® ELP). The second solubilizer may        be a polyethoxylated sorbitan monooleate (e.g. that sold under        the trade name Tween® 80).

The formulation may comprise:

-   -   from 0.5% to 6% by volume DMA;    -   from 0.5% to 6% by volume a first solubilizer;    -   from 0.2% to 4% by volume a second solubilizer;    -   from 85% to 99% by volume aqueous vehicle; and

from 2.0 mg to 12.0 mg (e.g. from 2.0 mg to 10.0 mg) per mLgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

The formulations described in the previous four paragraphs, in which thepolar aprotic solvent (e.g. DMA) is present as a minor component, can beused, for example, for administeringgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in the form of the(S)-phosphate epimer in substantially diastereomerically pure form. Theycan also be used for administering a mixture of R and S epimers or the Repimer. The formulations described in these paragraphs will typicallyhave been prepared by diluting a concentrated polar aprotic solvent(e.g. DMA) formulation or concentrated polar aprotic solvent (e.g. DMA)and solubilizer formulation with the aqueous vehicle up to 48 hoursprior to administration. The resulting formulations may be administeredthrough a peripheral vein.

While the formulations of the invention are preferably for parenteraladministration, certain embodiments of the invention may also beadministered orally.

In a second aspect of the invention is provided a pharmaceuticalformulation comprising:

-   -   gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate;    -   a polar aprotic solvent (e.g. DMA); and    -   optionally one or more pharmaceutically acceptable excipients;        wherein the formulation is for medical use.

In a third aspect of the invention is provided a pharmaceuticalformulation comprising:

-   -   gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate;    -   a polar aprotic solvent (e.g. DMA); and    -   optionally one or more pharmaceutically acceptable excipients;        wherein the formulation is for use in treating cancer.

In a fourth aspect of the invention is provided a method of treatingcancer, the method comprising administering to a subject in need thereofa pharmaceutical formulation comprising:

-   -   gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate;    -   a polar aprotic solvent (e.g. DMA); and    -   optionally one or more pharmaceutically acceptable excipients.

The method may comprise the steps of;

-   -   diluting a solution comprising        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, a polar        aprotic solvent (e.g. DMA) and optionally one or more        pharmaceutically acceptable excipients with an aqueous vehicle        to provide a formulation for infusion or injection; and    -   administering the formulation for infusion or injection to the        subject by infusion or injection.

The method may comprise the steps of;

-   -   diluting a first solution comprising        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate and a polar        aprotic solvent (e.g. DMA) and optionally an aqueous vehicle        with a second solution comprising a polar aprotic solvent (e.g.        DMA) and one or more solubilizers to form a third solution;    -   diluting the third solution with an aqueous vehicle to provide a        formulation for infusion or injection; and    -   administering the formulation for infusion or injection to the        subject by infusion or injection.

The second formulation may comprise more than one solubilizer.Typically, the second formulation will not comprise an active.

The or each dilution may be by a predetermined amount.

The starting solution may be a formulation of the first aspect.Likewise, the formulation for infusion or injection may be a formulationof the first aspect. It may be that the administration step is carriedout up to 48 hours (e.g. up to 12 or 2 hours) after the dilution step,e.g. the first or second dilution step.

The cancer may be a cancer selected from: pancreatic cancer, breastcancer, ovarian cancer, bladder cancer, colorectal cancer, lung cancer,bladder cancer, prostate cancer, cholangiocarcinoma, renal cancer,cervical cancer, thymic cancer, a cancer of an unknown primary origin,lymphoma or leukaemia.

The method may comprise:

a flushing a central line intravenous administration device with a firstportion of a first formulation, the first formulation comprising:

-   -   from 30% to 95% by volume DMA;    -   from 5% to 50% by volume aqueous vehicle; and        administering a second formulation to the patient via the        administration device, the second formulation comprising:    -   from 30% to 95% by volume DMA;    -   from 5% to 50% by volume aqueous vehicle; and        -   from 100 mg to 400 mg (e.g. from 100 mg to 300 mg) per mL            gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate; and

optionally flushing the administration device with a second portion ofthe first formulation. Typically, the first formulation will notcomprise an active. In a fifth aspect of the invention is provided amethod of preparing a pharmaceutical formulation ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate for infusion orinjection, the method comprising:

-   -   diluting a solution comprising        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, a polar        aprotic solvent (e.g. DMA) and optionally one or more        pharmaceutically acceptable excipients with an aqueous vehicle        to provide the formulation for infusion or injection.

The dilution may be by a predetermined amount.

The starting solution may be a formulation of the first aspect.Likewise, the formulation for infusion or injection may be a formulationof the first aspect. It may be that the administration step is carriedout up to 48 hours (e.g. up to 12 or 2 hours) after the dilution step.

The aqueous vehicle may be selected from saline (e.g. 0.9% saline or0.45% saline), glucose solution and water for infusion (WFI).Preferably, the aqueous vehicle is WFI. The use of WFI provides aformulation which is substantially isotonic with blood.

The aqueous vehicle may comprise one or more pharmaceutically acceptablesolubilizers (also known as a surfactants), e.g. a pharmaceuticallyacceptable non-ionic solubilizer. An exemplary solubilizer ispolyoxyethylene (20) sorbitan monooleate (marketed as Tween® 80).

In a sixth aspect of the invention is provided a method of preparing apharmaceutical formulation ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, the methodcomprising:

-   -   dissolving gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in        a polar aprotic solvent (e.g. DMA) to form a solution;    -   adding one or more further pharmaceutical excipients to the        solution to form a pharmaceutical formulation of        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

The inventors have discovered that a more efficient process arises frompredissolving the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate ina polar aprotic solvent (e.g. DMA) and then adding the requiredexcipients, e.g. solubilizers.

The one or more pharmaceutical excipients may include a solubilizer.

In a seventh aspect of the present invention is provided apharmaceutical formulation comprisinggemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate, or apharmaceutically acceptable salt or solvate thereof, and at least onepharmaceutically acceptable excipient. Preferably, thegemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate is insubstantially diastereoisomerically pure form.

The formulation may be for parenteral, e.g. for intravenous,subcutaneous or intramuscular administration. Preferably, theformulation is for intravenous administration.

The formulation may be an aqueous formulation which optionally alsocomprises a polar organic solvent. In the case of parenteral (e.g.intravenous) administration, the formulation preferably also comprises apolar organic solvent. The formulation may comprise DMSO or NMP.

The formulation may also comprise a cyclodextrin.

In a eighth aspect of the present invention is provided a pharmaceuticalformulation comprisinggemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate, or apharmaceutically acceptable salt or solvate thereof, and at least onepharmaceutically acceptable excipient. Preferably, thegemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate is insubstantially diastereoisomerically pure form.

In a ninth aspect of the invention is provided a kit, the kitcomprising:

-   -   a first formulation comprising:        -   from 30% to 95% by volume DMA;    -   from 5% to 50% by volume aqueous vehicle; and        a second formulation comprising:    -   from 30% to 95% by volume DMA;    -   from 5% to 50% by volume aqueous vehicle; and    -   from 100 mg to 400 mg (e.g. from 100 mg to 300 mg) per mL        gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate.

The first formulation will typically not comprise an active. Thus, itwill typically not comprisegemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The firstformulation may be provided in two separate vessels or in a singlevessel.

The kit of the ninth aspect of the invention is useful for theintravenous administration ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate via a central line.The central line is flushed with the first formulation prior toadministration of the second formulation. This mitigates the risk ofprecipitation of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate inor at the entrance to the intravenous administration apparatus, i.e. thecentral line, by avoiding the direct contact of the active formulationwith aqueous media (e.g. a saline flushing solution). The central linemay also be flushed with the first formulation after administration ofthe second formulation. This further prevents precipitation.

In a tenth aspect of the invention is provided a kit, the kitcomprising:

-   -   a first formulation comprising:        -   from 30% to 95% by volume DMA;        -   from 5% to 50% by volume aqueous vehicle; and        -   from 100 mg to 400 mg (e.g. from 100 mg to 300 mg) per mL            gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate; and    -   a second formulation comprising:        -   from 20% to 80% by volume DMA;        -   from 20% to 60% by volume a first solubilizer;        -   from 10% to 40% by volume a second solubilizer.

Typically the second formulation will not comprise any active. The kitis useful for the preparation of formulations suitable for peripheraladministration. The first formulation is diluted with the secondformulation up to 48 h, e.g. up to 24 h before administration to form athird formulation. The third formulation is further diluted with anaqueous vehicle before administration to the desired concentration toform the formulation which is used administered by infusion or injectionto the patient. In order to achieve formulations for peripheraladministration which are stable with respect to precipitation ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, it is typicallydesirable to include solubilizers. However, thegemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate can be prone todegradation in the presence of such solubilizers. Thus, a two stagedilution method is, in certain embodiments of the invention, thepreferable means by which formulations for peripheral administration areachieved.

DETAILED DESCRIPTION

Throughout this specification, the term S-epimer or S-diastereoisomerrefers to gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate.Likewise, throughout this specification, the term R-epimer orR-diastereoisomer refers togemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate.

The term ‘saline’ is intended to refer to an aqueous solution of sodiumchloride. Saline solutions of the present invention will typically besterile and will typically be at a concentration suitable for use inparenteral administration. Suitable concentrations are up to 2 w/v % orup to 1 w/v %. To optimise osmolarity different concentrations of salinecan be used in the formulations of the invention, e.g. 0.9% or 0.45%.

The formulations of the present invention can be used in the treatmentof the human body. They may be used in the treatment of the animal body.In particular, the compounds of the present invention can be used totreat commercial animals such as livestock. Alternatively, the compoundsof the present invention can be used to treat companion animals such ascats, dogs, etc.

The compounds in the formulations of the invention may be obtained,stored and/or administered in the form of a pharmaceutically acceptablesalt. Suitable pharmaceutically acceptable salts include, but are notlimited to, salts of pharmaceutically acceptable inorganic acids such ashydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic,and hydrobromic acids, or salts of pharmaceutically acceptable organicacids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic,benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic,stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic andvaleric acids. Suitable base salts are formed from bases which formnon-toxic salts. Examples include the aluminium, arginine, benzathine,calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium,meglumine, olamine, potassium, sodium, tromethamine and zinc salts.Hemisalts of acids and bases may also be formed, for example,hemisulfate, hemioxalate and hemicalcium salts. In certain embodiments,particularly those that apply to the s-epimer, the compound is in theform of a HCl salt or a hemioxalate salt. Preferably, the compound ofthe invention are not in the form of a salt, i.e. they are in the formof the free base/free acid.

For the above-mentioned formulations of the invention the dosageadministered will, of course, vary with the compound employed, theprecise mode of administration, the treatment desired and the disorderindicated. Dosage levels, dose frequency, and treatment durations ofcompounds of the invention are expected to differ depending on theformulation and clinical indication, age, and co-morbid medicalconditions of the patient. The size of the dose for therapeutic purposesof compounds of the invention will naturally vary according to thenature and severity of the conditions, the age and sex of the animal orpatient and the route of administration, according to well knownprinciples of medicine.

A pharmaceutical formulation typically takes the form of a compositionin which active compounds, or pharmaceutically acceptable salts thereof,are in association with a pharmaceutically acceptable adjuvant, diluentor carrier. One such pharmaceutically acceptable adjuvant, diluent orcarrier in the formulations of the invention is the polar aproticsolvent. Conventional procedures for the selection and preparation ofsuitable pharmaceutical formulations are described in, for example,“Pharmaceuticals—The Science of Dosage Form Designs”, M. E. Aulton,Churchill Livingstone, 1988.

The formulations may be suitable for topical application (e.g. to theskin or bladder), for oral administration or for parenteral (e.g.intravenous administration).

Any solvents used in pharmaceutical formulations of the invention shouldbe pharmaceutical grade, by which it is meant that they have an impurityprofile which renders them suitable for administration (e.g. intravenousadministration) to humans.

For oral administration the formulations of the invention may comprisethe active compound admixed with an adjuvant or a carrier, for example,lactose, saccharose, sorbitol, mannitol; a starch, for example, potatostarch, corn starch or amylopectin; a cellulose derivative; a binder,for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, forexample, magnesium stearate, calcium stearate, polyethylene glycol, awax, paraffin, and the like, and then compressed into tablets. If coatedtablets are required, the cores, prepared as described above, may becoated with a concentrated sugar solution which may contain, forexample, gum arabic, gelatine, talcum and titanium dioxide.Alternatively, the tablet may be coated with a suitable polymerdissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the active compounds maybe admixed with, for example, a vegetable oil or polyethylene glycol.Hard gelatine capsules may contain granules of the compound using eitherthe above-mentioned excipients for tablets. Also liquid or semisolidformulations of the active compounds may be filled into hard gelatinecapsules.

Liquid preparations for oral application may be in the form of syrups orsuspensions, for example, solutions containing the compound of theinvention, the balance being sugar and a mixture of ethanol, water,glycerol and propylene glycol. Optionally such liquid preparations maycontain colouring agents, flavouring agents, sweetening agents (such assaccharine), preservative agents and/or carboxymethylcellulose as athickening agent or other excipients known to those skilled in art.

Preferably, however the formulations of the invention are for parenteral(e.g. intravenous) administration or for dilution to form a formulationfor parenteral (e.g. intravenous) administration. For parenteral (e.g.intravenous) administration the active compounds may be administered asa sterile aqueous or oily solution. Preferably, the active compounds areadministered as a sterile aqueous solution.

The pharmaceutical composition of the invention will preferably comprisefrom 0.05 to 99% w (percent by weight)gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, more preferably from0.05 to 80% w gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, stillmore preferably from 0.10 to 70% wgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, and even morepreferably from 0.10 to 50% wgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, all percentages byweight being based on total composition.

Cyclodextrins have been shown to find wide application in drug delivery(Rasheed et al, Sci. Pharm., 2008, 76, 567-598). Cyclodextrins are afamily of cyclic oligosaccharides. They act as a ‘molecular cage’ whichencapsulates drug molecules and alters properties of those drugmolecules such as solubility. Cyclodextrins comprise (α-1,4)-linkedα-D-glucopyranose units. Cyclodextrins may contains 6, 7 or 8glucopyranose units (designated α-, β- and γ-cyclodextrinsrespectively). Cyclodextrins used in pharmaceutical formulations areoften β-cyclodextrins. The pendant hydroxyl groups can be alkylated witha C₁-C₈ substituted or unsubstituted alkyl group. Examples ofcyclodextrins are α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,2-hydroxypropyl-β-cyclodextrin (HP-β-CD), sulfobutylether β-cyclodextrinsodium salt, partially methylated β-cyclodextrin. The formulations ofthe invention may also comprise at least one cyclodextrin.

The present invention also includes formulations of all pharmaceuticallyacceptable isotopically-labelled forms of compound wherein one or moreatoms are replaced by atoms having the same atomic number, but an atomicmass or mass number different from the atomic mass or mass number of thepredominant isotope usually found in nature.

Examples of isotopes suitable for inclusion in the compounds of theinvention include isotopes of hydrogen, such as ²H and ³H, carbon, suchas ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F,iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen,such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulphur, such as³⁵S.

Certain isotopically-labelled compounds, for example, thoseincorporating a radioactive isotope, are useful in drug and/or substratetissue distribution studies. The radioactive isotopes tritium, i.e. ³H,and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose inview of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Isotopically-labelled compounds can generally be prepared byconventional techniques known to those skilled in the art or byprocesses analogous to those described using an appropriateisotopically-labelled reagent in place of the non-labelled reagentpreviously employed.

The method of treatment or the formulation for use in the treatment ofcancer, lymphoma or leukemia may involve, in addition to theformulations of the invention, conventional surgery or radiotherapy orchemotherapy. Such chemotherapy may include the administration of one ormore other active agents.

Where a further active agent is administered as part of a method oftreatment of the invention, such combination treatment may be achievedby way of the simultaneous, sequential or separate dosing of theindividual components of the treatment. Such combination products employthe compounds of this invention within a therapeutically effectivedosage range described hereinbefore and the one or more otherpharmaceutically-active agent(s) within its approved dosage range.

Thus, the pharmaceutical formulations of the invention may compriseanother active agent.

The one or more other active agents may be one or more of the followingcategories of anti-tumor agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof,such as alkylating agents (for example cyclophosphamide, nitrogenmustard, bendamustin, melphalan, chlorambucil, busulphan, temozolamideand nitrosoureas); antimetabolites (for example gemcitabine andantifolates such as fluoropyrimidines like 5-fluorouracil and tegafur,raltitrexed, methotrexate, pemetrexed, cytosine arabinoside, andhydroxyurea); antibiotics (for example anthracyclines like adriamycin,bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere and polokinase inhibitors); proteasomeinhibitors, for example carfilzomib and bortezomib; interferon therapy;and topoisomerase inhibitors (for example epipodophyllotoxins likeetoposide and teniposide, amsacrine, topotecan, mitoxantrone andcamptothecin);(ii) cytostatic agents such as antiestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5α-reductase suchas finasteride;(iii) anti-invasion agents, for example dasatinib and bosutinib(SKI-606), and metalloproteinase inhibitors, inhibitors of urokinaseplasminogen activator receptor function or antibodies to Heparanase;(iv) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies,for example the anti-erbB2 antibody trastuzumab [Herceptin™], theanti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab,tyrosine kinase inhibitors, for example inhibitors of the epidermalgrowth factor family (for example EGFR family tyrosine kinase inhibitorssuch as gefitinib, erlotinib and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib);inhibitors of the hepatocyte growth factor family; inhibitors of theinsulin growth factor family; modulators of protein regulators of cellapoptosis (for example Bcl-2 inhibitors); inhibitors of theplatelet-derived growth factor family such as imatinib and/or nilotinib(AMN107); inhibitors of serine/threonine kinases (for example Ras/Rafsignalling inhibitors such as farnesyl transferase inhibitors, forexample sorafenib, tipifarnib and lonafarnib), inhibitors of cellsignalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinaseinhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinaseinhibitors, IGF receptor, kinase inhibitors; aurora kinase inhibitorsand cyclin dependent kinase inhibitors such as CDK2 and/or CDK4inhibitors;(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, [for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin™);thalidomide; lenalidomide; and for example, a VEGF receptor tyrosinekinase inhibitor such as vandetanib, vatalanib, sunitinib, axitinib andpazopanib;(vi) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2;(vii) immunotherapy approaches, including for example antibody therapysuch as alemtuzumab, rituximab, ibritumomab tiuxetan (Zevalin®) andofatumumab; interferons such as interferon α; interleukins such as IL-2(aldesleukin); interleukin inhibitors for example IRAK4 inhibitors;cancer vaccines including prophylactic and treatment vaccines such asHPV vaccines, for example Gardasil, Cervarix, Oncophage and Sipuleucel-T(Provenge); and toll-like receptor modulators for example TLR-7 or TLR-9agonists;(viii) cytotoxic agents for example fludaribine (fludara), cladribine,pentostatin (Nipent™);(ix) steroids such as corticosteroids, including glucocorticoids andmineralocorticoids, for example aclometasone, aclometasone dipropionate,aldosterone, amcinonide, beclomethasone, beclomethasone dipropionate,betamethasone, betamethasone dipropionate, betamethasone sodiumphosphate, betamethasone valerate, budesonide, clobetasone, clobetasonebutyrate, clobetasol propionate, cloprednol, cortisone, cortisoneacetate, cortivazol, deoxycortone, desonide, desoximetasone,dexamethasone, dexamethasone sodium phosphate, dexamethasoneisonicotinate, difluorocortolone, fluclorolone, flumethasone,flunisolide, fluocinolone, fluocinolone acetonide, fluocinonide,fluocortin butyl, fluorocortisone, fluorocortolone, fluocortolonecaproate, fluocortolone pivalate, fluorometholone, fluprednidene,fluprednidene acetate, flurandrenolone, fluticasone, fluticasonepropionate, halcinonide, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone valerate, icomethasone, icomethasone enbutate,meprednisone, methylprednisolone, mometasone paramethasone, mometasonefuroate monohydrate, prednicarbate, prednisolone, prednisone,tixocortol, tixocortol pivalate, triamcinolone, triamcinolone acetonide,triamcinolone alcohol and their respective pharmaceutically acceptablederivatives. A combination of steroids may be used, for example acombination of two or more steroids mentioned in this paragraph;(x) targeted therapies, for example PI3Kd inhibitors, for exampleidelalisib and perifosine; or compounds that inhibit PD-1, PD-L1 and CART.

The one or more other active agents may also be antibiotic.

As an illustrative example, a diastereomeric mixture ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate can be preparedaccording to the synthetic methods described in WO2005/012327 or thosedescribed in ‘Application of Pro Tide Technology to Gemcitabine: ASuccessful Approach to Overcome th Key Cancer Resistance MechanismsLeads to a New Agent (NUC-1031) in Clinical Development’; Slusarczyk etall; J. Med. Chem.; 2014, 57, 1531-1542.

The (R) and (S) isomers ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate can be separated byHPLC under the following conditions:

Equipment: Agilent 1200™ series with DAD detector

Flow rate: 1.0 mL/min

Column: Chiralpak AD™; 250×4.6 mm ID (normal phase)

Temperature: ambient

Particle size: 20 μm

Feed: dissolved in MeOH; 10 g/L

Solvent: n-heptane/IPA 10 →50% IPA

The (S)-epimer eluted at 8.6 minutes and the (R)-epimer eluted at 10.3minutes.

The individual isomers can be characterised using the followingcharacterisation methods: Proton (¹H), carbon (¹³C), phosphorus (³¹P)and fluorine (¹⁹F) NMR spectra were recorded on a Bruker Avance 500spectrometer at 25° C. Spectra were auto-calibrated to the deuteratedsolvent peak and all ¹³C NMR and ³¹P NMR were proton-decoupled. Thepurity of final compounds was verified to be >95% by HPLC analysis usingVarian Polaris C18-A (10 μM) as an analytic column with a gradientelution of H₂O/MeOH from 100/0 to 0/100 in 35 min. The HPLC analysis wasconducted by Varian Prostar (LC Workstation-Varian prostar 335 LCdetector).

2′-Deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzyloxy-L-alaninyl)]-(S)-phosphate3

(ES+) m/z, found: (M+Na⁺) 603.14. C₂₅H₂₇F₂N₄O₈NaP required: (M⁺) 580.47.

³¹P NMR (202 MHz, MeOD): δ_(P) 3.66

¹H NMR (500 MHz, MeOD): δ_(H) 7.58 (d, J=7.5 Hz, 1H, H-6), 7.38-7.32 (m,7H, ArH), 7.26-7.20 (m, 3H, ArH), 6.24 (t, J=7.5 Hz, 1H, H-1′), 5.84 (d,J=7.5 Hz, 1H, H-5), 5.20 (AB system, J_(AB)=12.0 Hz, 2H, OCH₂Ph),4.46-4.43 (m, 1H, H-5′), 4.36-4.31 (m, 1H, H-5′), 4.25-4.19 (m, 1H,H-3′), 4.07-4.00 (m, 2H, H-4′, CHCH₃), 1.38 (d, J=7.2 Hz, 3H, CHCH₃).

¹⁹F NMR (470 MHz, MeOD): δ_(F) −118.0 (d, J=241 Hz, F), −120.24 (broadd, J=241 Hz, F).

¹³C NMR (125 MHz, MeOD): δ_(C) 174.61 (d, ³J_(C—P)=5.0 Hz, C═O, ester),167.63 (C—NH₂), 157.74 (C═O base), 152.10 (d, ²J_(C—P)=7.0 Hz, C—Ar),142.40 (CH-base), 137.22 (C—Ar), 130.90, 129.63, 129.39, 129.32, 126.32(CH—Ar), 124.51 (d, ¹J_(C—F)=257 Hz, CF₂), 121.47, 121.43 (CH—Ar), 96.67(CH-base), 85.92 (broad signal, C-1′), 80.31 (C-4′), 71.27 (apparent t,²J_(C—F)=23.7 Hz, C-3′), 68.03 (OCH₂Ph), 65.73 (d, ²J_(C—P)=5.30 Hz,C-5′), 51.66 (CHCH₃), 20.42 (d, ³J_(C—P)=6.25 Hz, CHCH₃).

Reverse HPLC, eluting with H₂O/MeOH from 100/0 to 0/100 in 35 min,showed one peak of diastereoisomer with t_(R)=22.53 min.

2′-deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzyloxy-L-alaninyl)]-(R)-phosphate4

(ES+) m/z, found: (M+Na⁺) 603.14. C₂₅H₂₇F₂N₄O₈NaP required: (M⁺) 580.47.

³¹P NMR (202 MHz, MeOD): δ_(P) 3.83

¹H NMR (500 MHz, MeOD): δ_(H) 7.56 (d, J=7.5 Hz, 1H, H-6), 7.38-7.31 (m,7H, ArH), 7.23-7.19 (m, 3H, ArH), 6.26 (t, J=7.5 Hz, 1H, H-1′), 5.88 (d,J=7.5 Hz, 1H, H-5), 5.20 (s, 2H, OCH₂Ph), 4.49-4.46 (m, 1H, H-5′),4.38-4.34 (m, 1H, H-5′), 4.23-4.17 (m, 1H, H-3′), 4.07-4.01 (m, 2H,H-4′, CHCH₃), 1.38 (d, J=7.2 Hz, 3H, CHCH₃).

¹⁹F NMR (470 MHz, MeOD): δ_(F) −118.3 (d, J=241 Hz, F), −120.38 (broadd, J=241 Hz, F).

¹³C NMR (125 MHz, MeOD): δ_(C) 174.65 (d, ³J_(C—P)=5.0 Hz, C═O, ester),167.65 (C—NH₂), 157.75 (C═O base), 152.10 (d, ²J_(C—P)=7.0 Hz, C—Ar),142.28 (CH-base), 137.50 (C—Ar), 130.86, 129.63, 129.40, 129.32, 126.31(CH—Ar), 124.50 (d, ¹J_(C—F)=257 Hz, CF₂), 121.44, 121.40 (CH—Ar), 96.67(CH-base), 85.90 (broad signal, C-1′), 80.27 (C-4′), 71.30 (apparent t,²J_(C—F)=23.7 Hz, C-3′), 68.02 (OCH₂Ph), 65.50 (C-5′), 51.83 (CHCH₃),20.22 (d, ³J_(C—F)=7.5 Hz, CHCH₃).

Reverse HPLC, eluting with H₂O/MeOH from 100/0 to 0/100 in 35 min,showed one peak of diastereoisomer with t_(R)=21.87 min

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or process so disclosed, may be combined inany combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive. The invention is notrestricted to the details of any foregoing embodiments. The inventionextends to any novel one, or any novel combination, of the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings), or to any novel one, or any novel combination,of the steps of any method or process so disclosed.

The readers attention is directed to all papers and documents which arefiled concurrently with or previous to this specification in connectionwith this application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

The following abbreviations are used in this specification:

API—active pharmaceutical ingredient, i.e.gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate

DMA - dimethylacetamide DMF - N,N-dimethylformamide DMSO-dimethylsulfoxide IPA - isopropyl alcohol NMP - N-methylpyrroldinonePEG - polyethylene glycol

Example 1—Developing a First Generation Formulation

Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2) wasobtained as a mixture of phosphate diastereoisomers by the methoddescribed in WO2005/012327.

The experiments of Example 1 were all conducted using NUC-1031 as amixture of phosphate diastereoisomers.

The solubility of NUC-1031 was determined in a range of pharmaceuticallyacceptable solvent systems. The protocol adopted was as follows:

A small volume, 1-2 mL, of each solvent system was prepared and a weightof the compound in question was added. The solutions were stirred forapproximately 4 hours and then 0.45 μL membrane filtered. Theconcentration of the compound in question in the filtrate was thendetermined by HPLC assay.

Based on the gemcitabine dosage schedule used in the treatment ofpancreatic cancer, the molecular weight adjusted dose of NUC-1031 wouldbe about 3200 mg, given as an infusion once weekly. As an indication ofthe level of solubility required, taking a notional target of a 500 mLinfusion volume, the required solubility of the NUC-1031 would be >6mg/ml in the infusion fluid. However, this solubility level is just anindication and lower solubilities can still provide effective therapies.

TABLE 1 shows the solubility of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate 2 in a range of solvents suitable for intravenousadministration. Solvent Appearance Assay(mg/ml) Ethanol Solubilisedquickly, after 30 minutes precipitated out to white paste Glycerol APIevident Propylene glycol Precipitation evident 371 after 30 minutes PEG400 Precipitation evident 385 after 120 minutes NMP Clear solution >207DMSO Clear solution >217 DMA Clear solution >656

DMSO, DMA and NMP, all of which are polar aprotic solvents, providedstable solutions.

After dilution 1:1 with water or saline NMP and DMA did not show anyevidence of precipitation. Appendix 1 shows the solubility of NUC-1031in a range of solvents on dilution. DMA provided sufficient solubilityto administer the required dose

TABLE 2 shows the solubility of NUC-1031 in a range of solvents ondilution Evidence of further precipitation Solvent: NUC-1031 mg/mLRecovery on storage Solvent, quantity Saline HPLC assay from of filtrateat of NUC-1031 (0.9%) Appearance filtrate theoretical RT >24 h PEG 400,91.2 mg/mL 1:1  Clear n/a n/a Yes solution PEG 400, 91.2 mg/mL 1:2 Precipitation 16.2 53% Yes evident PEG 400, 91.2 mg/mL 1:2* Slightlyturbid 18.8 62% Yes solution PEG 400, 45.6 mg/mL  1:1.5* Clear n/a n/aYes solution PEG 400, 45.6 mg/mL 1:2* Clear n/a n/a Yes solution PEG400, 45.6 mg/mL  1:2.5* Precipitation, 10.5 80% Yes solution alsoprecipitated after filtration DMA 92.5 mg/mL      1:1 glucose Clear 47.3102%  No solution DMA 92.5 mg/mL      1:2 glucose Slightly turbid 29.796% Yes solution PEG 400 87.7 mg/mL      1:1 glucose Slightly turbid46.1 105%  Yes solution PEG 400 87.7 mg/mL      1:2 glucose Turbid 17.460% No solution/ precipitation NMP 115.0 mg/mL    1:1 saline Slightlyturbid 60.0 104%  No solution NMP 115.0 mg/mL    1:2 saline Slightlyturbid 40.5 106%  Yes solution NMP 115.0 mg/mL      1:1 glucose Slightlyturbid 58.5 102%  No solution NMP 115.0 mg/mL      1:2 glucose Slightlyturbid 39.6 103%  Yes solution DMA 91.6 mg/mL 1:1  Clear 47.0 103% solution DMA 91.6 mg/mL 1:2  Slightly turbid 30.2 99% solution DMA 91.6%mg/mL 1:3  Precipitation 14.8 65% observed DMA 91.6 mg/mL 1:2* Initiallyclear 30.9 101%  >30 min slight precipitation DMA 91.6 mg/mL 1:3*Precipitation 15.2 66% evident DMA 73.3 mg/mL 1:3* Precipitation 14.780% evident DMA 55.0 mg/mL 1:3* Slightly turbid 13.9 101%  solution DMA45.8 mg/mL 1:3* Clear 11.5 100%  solution DMA 45.8 mg/mL  1:3.5* Clearn/a n/a solution DMA 45.8 mg/mL 1:4* Initially clear  8.4 92%precipitates >30 min, stirring precipitate dissolves DMA 45.8 mg/mL 1:4.5* Slightly turbid  7.2 87% solution *0.9% saline containing 0.13%Tween 80Effects of Dilution on DMA Solubility

Table 2 gives the effect of aqueous dilution on DMA solubility

TABLE 2 Solution Assay (mg/ml) Precipitation > 24 hours 100% DMA 592 No95:5 DMA: 518 No 0.9% Saline 90:10 DMA: 483 No 0.9% Saline 80:20 DMA:386 Yes 0.9% Saline 70:30 DMA: 339 Yes 0.9% Saline 60:40 DMA: 293 Yes0.9% Saline 50:50 DMA: 66 Yes 0.9% Saline

These DMA solutions were further evaluated for physical stability over alonger time and the results are given in Table 2a

TABLE 2a Solution in Assay Precipitation 0.9% Saline (mg/ml) (2 weeks)80:20 DMA 304 Yes 80:20 DMA 272 No 80:20 DMA 315 Yes 80:20 DMA 270 Yes85:15 DMA 338 No

Following the experiments described above a formulation of 250 mgNUC-1031 in a 80:20 DMA:0.9% saline solution in a 5 ml vial was used inclinical testing. The formulation provided a successful treatment in theclinical study but needed to be administered by a central line becauseof pain on injection.

A formulation allowing administration by peripheral veins was thensought.

Example 2

The experiments of Examples 2 to 6 were all conducted using the(S)-epimer of NUC-1031.

Compounding

NUC-1031 was compounded into nine different formulations using DMA and aco-excipient as described in Table 3.

TABLE 3 NUC-1031 Formulations NUC-1031 DMA Co-excipient FormulationWeight Volume Co-excipient Volume A 1 g 3 mL Kolliphor ® EL 7 mL B 1 g 4mL Kolliphor ® EL 6 mL C 1 g 3 mL Kolliphor ® ELP 7 mL D 1 g 4 mLKolliphor ® ELP 6 mL E 1 g 3 mL Kolliphor ® HS15 7 mL F 1 g 4 mLKolliphor ® HS15 6 mL G 1 g 4 mL PEG 400 6 mL H 1 g 4 mL PEG 300 6 mL I1 g 4 mL Polyethylene 6 mL Glycol

The API was compounded using the following method:

-   -   1. The DMA was added to NUC-1031 in a glass scintillation vial.        Instant dissolution of the API was observed.    -   2. The co-excipient was added second and briefly mixed (less        than a minute) using a vortex mixer (Whirlmixer, Fisher brand).

It was found that this provided a more efficient method of compoundingthe API than dissolving NUC-1031 in a mixture of the DMA and theco-excipient. Dissolving the NUC-1031 in the mixture does still providethe compounded API but the process is less efficient.

All of the formulations were clear solutions which remained stable (byeye) for several days (>7 days).

It was observed that the API contributes to the formulation volume. Atypical formulation in this study has a volume of 10.6-10.7 mL (APIconcentration 93-94 mg/mL).

Example 3—Infusion Solution Studies

The solubility of the NUC-1031 formulations in infusion solutions wasinvestigated.

In the clinic it is intended to solubilise 2 g of API in 500 mL ofinfusion solution (4 mg/mL). The formulations described above werediluted to generate an infusion solution with a slightly higher APIconcentration (4.6-4.7 mg/mL) to represent a worst case scenario. Theresults are shown in Table 4.

TABLE 4 Solubility of NUC-1031 Formulations in Infusion Solutions (p =precipitate; c = clear solution) Infusion T = 0 T = 2 T = 4.5 T = 7 T =24 Formulation Solution hours hours hours hours hours A 0.45% saline c cc c p B WFI c c p p p C 0.45% saline c c c p p D WFI c c p p p E 0.45%saline c c c c p F WFI c c c c c G WFI p n/a n/a n/a n/a H WFI p n/a n/an/a n/a 1 WFI p n/a n/a n/a n/a

Formulations B and F were selected for infusion bag studies.

Example 4—Infusion Bad Studies

Formulations B and F (5 mL of each) were injected into 100 mL WFI BaxterViaflo® bags. Viaflo® bags are manufactured from a PVC free plastic.This eliminates the risk of leaching toxic phthalate compounds.

TABLE 5 Solubility of Formulations B and F in WFI Infusion Bags (p =precipitate; c = clear solution) Infusion T = 2 T = 24 FormulationSolution T = 0 hours hours Formulation B: WFI c c P API - 1 g, DMA 4 mL,Kolliphor ® EL - 6 mL Formulation F: WFI c c P API - 1 g, DMA 4 mL,Kolliphor ® HS15-6 mL

The above results show that formulations comprising DMA can be generatedwhich, upon dilution with an aqueous vehicle, are capable of remainingstable for long enough to be administered to a patient. The formulationscan be diluted until the DMA is a relatively minor component (1-2%),with the majority of the remainder of the solvent being water withoutgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate precipitating out ofsolution.

Example 5—Further Formulation Stability Studies

A range of further formulations of the (S)-isomer ofgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate were prepared andinvestigated (Table 6).

TABLE 6 Further (S)-Isomer formulations Target API API ConcentrationWeight* Formulation Formulation (mg/mL) (g) Volume (mL) Excipients J 751.90 25 30% DMA, 70% Kolliphor ® ELP K 75 1.90 25 40% DMA, 60%Kolliphor ® ELP L 75 1.89 25 50% DMA, 50% Kolliphor ® ELP M 75 1.89 2550% DMA, 50% Tween ® 80 N 100 2.53 25 30% DMA, 70% Kolliphor ® ELP O 1002.54 25 40% DMA, 60% Kolliphor ® ELP P 100 2.54 25 50% DMA, 50%Kolliphor ® ELP Q 100 2.53 25 50% DMA, 50% Tween ® 80 *The actual APweight factored in the potency 99.1% of the API

For each formulation the API was initially solubilised in DMA and thenmade up to volume in the volumetric flask with either Kolliphor® ELP orTween® 80. The Kolliphor® ELP was melted by applying the minimum amountof heat required to achieve melting (50° C. oven, 10 minutes).

Filtration and Filling

The formulations were filtered manually through a syringe filter into 2mL clear glass vials.

The formulations afforded a back pressure during filtration that made itphysically difficult to pass the solution through a given filter andwhich contributed to sample loss. The greater the concentration ofKolliphor® ELP in the formulation the greater the back pressureexperienced during filtration was.

The head space of the filled vials was flushed with nitrogen prior tosealing with a 13 mm West stopper and crimping with an aluminiumoverseal.

All of the vials were stored at 2-8° C. for 3 days prior to T=0 testingand putting into stability. No precipitate formation or gelling wasobserved in any of the vials.

Stability

For each formulation four vials were assessed for stability at 25° C.and four vials at 2-8° C.

Appearance—Batches 1-3 and 5-7 conformed to the description “clearcolourless solution, free from visible particulates” at T=0 and 1 monthat all storage conditions. Batches 4 and 8 conformed to the description“clear yellow solution, free from visible particulates” at T=0 and 1month at all storage conditions.

Assay and Related Substances—Samples were analysed using the assay andrelated substances method ADP173 vs. 04 for NUC-1031. For the 100 mg/mLsamples 200 μl was transferred to a 20 mL volumetric flask usingpositive displacement pipette and diluted to volume with diluent. Forthe 75 mg/mL samples 250 μl was transferred to a 20 mL volumetric flaskusing positive displacement pipette and diluted to volume with diluent.

TABLE 7 Assay 2-8° C. Assay Assay (mg/mL) (mg/mL) 2-8° C. Formulation T= 0 T = 1 m J 77.43 73.96 K 78.56 74.82 L 75.59 75.90 M 74.21 71.73 N108.27 101.05 O 95.09 97.97 P 96.48 95.75 Q 94.95 73.90

TABLE 8 Assay 25° C./60% relative humidity Assay (mg/mL) Assay (mg/mL)25° C./60% Formulation T = 0 RH T = 1 m J 77.43 73.90 K 78.56 74.74 L75.59 75.94 M 74.21 64.80 N 108.27 103.76 O 95.09 98.51 P 96.48 97.70 Q94.95 89.05

The formulations were then diluted in 0.45% saline and the stability wasevaluated as indicated in Table 9.

TABLE 9 Stability of formulations in 0.45% saline API concentration in0.45% saline Osmolality Observation Observation Formulation Excipients(mg/mL) pH (mOsm/kg) T = 6 hours T = 24 hours J 30% DMA, 3 6.2 281 ClearClear 70% Kolliphor ® ELP solution solution K 40% DMA, 3 6.3 316 ClearClear 60% Kolliphor ® ELP solution solution L 50% DMA, 3 6.5 371 ClearClear 50% Kolliphor ® ELP solution solution M 50% DMA, 3 7.1 377 ClearClear 50% Tween ® 80 solution solution N 30% DMA, 5 6.3 292 ClearPrecipitate - 70% Kolliphor ® ELP solution small amount 0 40% DMA, 5 6.3458 Clear Precipitate - 60% Kolliphor ® ELP solution small amount P 50%DMA, 5 6.3 437 Clear Precipitate - 50% Kolliphor ® ELP solution largeamount Q 50% DMA, 5 7.0 471 Clear Solid gel 50% Tween ® 80 solution

The results indicate that the 75 mg/mL formulations (J-M) diluted to 3mg/mL in 0.45% saline are physically stable for 24 hours. The 100 mg/mLformulations (N-Q) diluted to 5 mg/mL in 0.45% saline are physicallystable up to 6 hours. Formulations L and O were evaluated on a differentday by a different operator and the same results were obtained.

Infusion Solution Evaluation

The long term stability of the formulations were evaluated by dilutingwith 0.45% saline after the formulations had been stored for 1 month asindicated in Table 10.

TABLE 10 Formulations in 0.45% saline T = 1 month API concentration in0.45% saline Observation Formulation Sample Excipients (mg/mL) T = 24hours J T = 1 month 30% DMA, 3 Clear solution 2-8° C. 70% Kolliphor ®ELP J T = 1 month 30% DMA, 3 Clear solution 25° C. 70% Kolliphor ® ELP KT = 1 month 40% DMA, 3 Clear solution 2-8° C. 60% Kolliphor ® ELP K T =1 month 40% DMA, 3 Clear solution 25° C. 60% Kolliphor ® ELP L T = 1month 50% DMA, 3 Clear solution 2-8° C. 50% Kolliphor ELP L T = 1 month50% DMA, 3 Clear solution 25° C. 50% Kolliphor ® ELP M T = 1 month 50%DMA, 3 Clear solution 2-8° C. 50% Tween ® 80 M T = 1 month 50% DMA, 3Clear solution 25° C. 50% Tween ® 80 N T = 1 month 30% DMA, 3 Clearsolution 2-8° C. 70% Kolliphor ® ELP N T = 1 month 30% DMA, 3 Clearsolution 25° C. 70% Kolliphor ® ELP O T = 1 month 40% DMA, 3 Clearsolution 2-8° C. 70% Kolliphor ® ELP O T = 1 month 40% DMA, 3 Clearsolution 25° C. 60% Kolliphor ® ELP P T = 1 month 50% DMA, 3 Clearsolution 2-8° C. 50% Kolliphor ® ELP P T = 1 month 50% DMA, 3 Clearsolution 25° C. 50% Kolliphor ® ELP Q T = 1 month 50% DMA, 3 Clearsolution 2-8° C. 50% Tween ® 80 Q T = 1 month 50% DMA, 3 Clear solution25° C. 50% Tween ® 80 [00138]

The results indicate that the 75 mg/mL formulations (J-M) and the 100mg/mL formulations (N-Q) which have been stored for 1 month and thendiluted to 3 mg/mL in 0.45% saline are physically stable after 24 hours.

The formulations that had been stored at 25° C. (for 2 months) and thatcontained Kolliphor ELP™ were evaluated in filtered 0.45% saline at anumber of concentrations as indicated in Table 11.

TABLE 11 NUC-1031 formulations in 0.45% saline, T = 2 months, 25° C. APIconcentration in 0.45% saline Observation Formulation Composition(mg/mL) T = 19 hours J 75 mg/mL API, 30% DMA, 3 Clear solution 70%Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution K75 mg/mL, 40% DMA, 3 Clear solution 60% Kolliphor ® ELP 3.5 Clearsolution 4 Clear solution 4.5 Clear solution L 75 mg/mL API, 50% DMA, 3Clear solution 50% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution4.5 Clear solution N 100 mg/mL API, 30% DMA, 3 Clear solution 70%Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution O100 mg/mL API, 40% DMA, 3 Clear solution 60% Kolliphor ® ELP 3.5 Clearsolution 4 Clear solution 4.5 Clear solution P 100 mg/mL API, 50% DMA, 3Clear solution 50% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution4.5 Clear solution

The results indicate that the formulations diluted in 0.45% saline arephysically stable up to a concentration of 4.5 mg/mL.

Example 7—Combinations of Solubilizers

Samples were prepared in which a combination of solubilizers waspresent.

First a 250 mg/mL solution of the S-epimer in DMA was prepared bydissolving the S-epimer in DMA. This was then diluted to a 100 mg/mLsolution by addition of the desired combination of solubilizers,according to Table 12.

Formulation Kolliphor ® ELP Kolliphor ® HS15 No DMA % % % Tween ® 80% 140 30 30 2 40 20 40 3 40 40 20 4 40 30 30 5 40 20 40 6 40 40 20 7 40 3030 8 40 20 40 9 40 40 20 10 40 10 20 30 11 40 10 30 20 12 40 20 10 30 1340 20 30 10 14 40 30 20 10 15 40 30 10 20 16 40 20 20 20

The formulations were each diluted in 0.45% saline (pH 5.9) to providesolutions that were 4 mg/mL, 6 mg/mL, 8 mg/mL and 10 mg/mL. Theappearance of the solution was checked after stirring and after 3 hours,6 hours and 24 hours of storage at ambient temperature. All solutions,including those at 10 mg/mL remained clear colourless solutions after 24hours. The 10 mg/mL solution of formulation 3 did however show somecloudiness and particulate formation after 26 hours. HPLC analysis ofthe other 10 mg/mL solutions showed that the concentration of the activein solution and the purity of the active remained at the expectedlevels.

Thus, the use of combinations of more than one solubilizer can allowstable solutions of NUC-1031 to be formed at higher concentrations.

Example 8

A preferred formulation system for formulating NUC-1031 is as follows:

A 250 mg/mL solution of NUC-1031 (the S-epimer, the R epimer or amixture thereof) is formed in an 80:20 (by volume) mixture of DMA and0.9% saline. This system is sufficiently stable for long term storageand transport of NUC-1031.

This formulation can be administered to patients intravenously via acentral line (e.g. a Hickman line, PICC line, Portacath). Theintravenous administration apparatus will typically be flushed with an80:20 (by volume) mixture of DMA and 0.9% saline both before and afteradministration of the formulation comprising NUC-1031. This helpsmitigate the risk of any potential precipitation of NUC-1031 in theintravenous administration apparatus on contact with the saline flush.

Alternatively, where intravenous administration into a peripheral veinis the preferred method of administration this first formulation is thendiluted to 100 mg/mL with a 40%:40%:20% mixture of DMA:Tween®80:Kolliphor® ELP (eg 6.9 mL of 250 mg/ml NUC-1031 in 80:20 DMA:0.9%saline is added to 10.35 mL of the DMA:Tween®80:Kolliphor® ELP diluent).The resultant (second) formulation has been shown to be stable for up to5 days for both the S-epimer and for a mixture of the R and S epimers.

The final administration formulation is then prepared by diluting thissecond formulation to the desired concentration with saline. Solutionsof a mixture of the R and S epimers at 4, 8 and 10 mg/mL have been shownto be stable (both to precipitation of NUC-1031 and to degradation ofNUC-1031) for 48 hours after dilution of this formulation in both 0.45%and 0.9% saline at a range of pHs (4.5, 6.0 and 7.0), providing themixtures were not stirred. The osmolarity of all of these solutions hasalso been shown to be acceptable for peripheral administration.

The invention claimed is:
 1. A pharmaceutical formulation comprising:from 10% to 60% by volume DMA; from 10% to 80% by volume solubilizer orsolubilizers; from 1% to 15% by volume aqueous vehicle; and from 50 mgto 150 mg per mL gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate,wherein each solubilizer is a non-ionic surfactant or a mixture thereof;and wherein the aqueous vehicle is selected from saline, glucosesolution and water for infusion (WFI).
 2. The pharmaceutical formulationaccording to claim 1, wherein the aqueous vehicle is saline.
 3. Thepharmaceutical formulation according to claim 1, wherein the aqueousvehicle is WFI.
 4. The pharmaceutical formulation according to claim 1,wherein each solubilizer is a polyethoxylated fatty acid or a mixturethereof.
 5. The pharmaceutical formulation according to claim 1, whereineach solubilizer is a polyoxyl castor oil or a mixture thereof.
 6. Thepharmaceutical formulation according to claim 1, whereingemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate is in the form ofthe (S)-phosphate epimer with a diastereoisomeric purity of greater than95%.
 7. The pharmaceutical formulation according to claim 1 for dilutionwith an aqueous vehicle to form a formulation for intravenousadministration.
 8. A method of preparing a pharmaceutical formulationaccording to claim 1, the method comprising: diluting a firstformulation with a second formulation; wherein the first formulationcomprises: from 200 mg to 300 mg per mLgemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate; from 70% to 90% byvolume dimethyl acetamide (DMA); and from 10% to 30% by volume aqueousvehicle; and the second formulation comprises: from 20% to 70% by volumeDMA; from 10% to 60% by volume a first solubilizer; and from 10% to 60%by volume a second solubilizer, wherein each solubilizer is a non-ionicsurfactant or a mixture thereof; and wherein the aqueous vehicle isselected from saline, glucose solution and water for infusion (WFI). 9.The method according to claim 8, whereingemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate is in the form ofthe (S)-phosphate epimer with a diastereoisomeric purity of greater than95%.
 10. A method of treating cancer, the method comprising: diluting asolution comprising: from 10% to 60% by volume DMA; from 30% to 80% byvolume solubilizer or solubilizers; from 1% to 15% by volume aqueousvehicle; and from 50 mg to 150 mg per mLgemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate, with an aqueousvehicle to provide a formulation for infusion or injection; andadministering the formulation for infusion or injection to the subjectby infusion or injection, wherein each solubilizer is a non-ionicsurfactant or a mixture thereof; and wherein the aqueous vehicle isselected from saline, glucose solution and water for infusion (WFI). 11.The method according to claim 10, whereingemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate is in the form ofthe (S)-phosphate epimer with a diastereoisomeric purity of greater than95%.